The genus flavivirus contains approximately 70 positive single-stranded RNA viruses, among which many major human pathogens are found, including Dengue virus (“DV”), West Nile (“WNV”), Yellow Fever virus (“YFV”), Japanese and tick-borne encephalitis viruses. YFV was the first flavivirus to be isolated in 1927, but historically, flavivirus-like diseases have been reported in the medical literature since at least 1780.
On of the most common and virulent flaviviruses is DV. DV threatens up to 2.5 billion people in 100 endemic countries. Up to 50 million infections occur annually with 500 000 cases of dengue haemorrhagic fever and 22,000 deaths mainly among children. Dengue has been classified by the World Health Organization (“WHO”) as a priority as it ranks as the most important mosquito-borne viral disease in the world. In the last 50 years, its incidence has increased 30-fold. Prior to 1970, only 9 countries had experienced cases of dengue haemorrhagic fever (“DHF”); since then the number has increased more than 4-fold and continues to rise.
WNV also has become much more wide-spread. In 1999, WNV was isolated for the first time in the Americas during an outbreak in New York City. By the end of 2002, WNV activity had been identified in 44 states of the United States and the District of Columbia. The 2002 WNV epidemic resulted in 4,156 reported human cases of WN disease including 2,942 meningoencephalitis cases and 284 deaths.
There have been previous attempts to generate a vaccine. For example, a live, attenuated virus of YFV (strain 17D) was developed in 1936 and has been used as a vaccine for over 400 million people. Unfortunately, the vaccine has not proved 100% successful since there are 200,000 estimated cases of yellow fever (with 30,000 deaths) per year worldwide, 90% of which in Africa. Chimeric live vaccines incorporating genes of either Japanese encephalitis, WNV, or Dengue in a YFV 17D vector are currently in development. However, a number of difficulties are associated with the conception of safe and efficient vaccines, such as vaccine purity, and immunogenic cross responses. That is why antiviral chemotherapy has a major role to play in the control of such diseases.
Since viral RNA polymerase is critical for replication of the virus and cannot be substituted by any other cellular polymerase, it is an excellent antiviral target. As a result, most of the more than 30 new antiviral agents, which have been developed and approved during the last 5 years, are directed against viral polymerases. They are mainly targeted against human immuno-deficiency virus, but drugs against hepatitis B and C, herpes simplex, varicella-zoster and influenza virus infections have also been made commercially available.
More than 50% of these antiviral agents are nucleoside analogues, in which the base, the ribose moiety or both have been modified. Nucleoside analogues can act as inhibitory ligands by binding to the template binding site within the polymerase active site and preventing the access of the viral RNA, or by binding to the nucleotide binding site, thus limiting the availability of the natural substrate for complementary strand synthesis. It is generally understood in the art that a nucleoside analogue may be a synthetic molecule that resembles a naturally occurring nucleoside, but lacks a bond site needed to link it to an adjacent nucleotide. Additionally, nucleoside analogues can also act as chain-terminators during DNA or RNA synthesis, by binding themselves as a substrate for the target polymerase, but preventing further chain elongation. Non-nucleoside analogues may bind to allosteric sites thus influencing the local conformation of the active site via long-range conformational changes of the polymerase's structure.
Another approach whereby many antiviral compounds have been discovered is by using cell cultures infected with the virus of interest. In such cases, addition of an antiviral compound protects the cells from infection, or inhibits virus growth. For this type of experiment, it is useful to identify a large number of antiviral compounds in an efficient manner. As such, another evolving mechanism to identify new antiviral agents through the high-throughput screening (“HTS”) of a large number of synthetic or natural compounds. This requires the development of an in vitro assay, which in turn requires large amounts of soluble and active protein.
When a high number of potentially antiviral compounds are tested by HTS, it is possible to identify antiviral compounds in an efficient manner. This approach has been used successfully for HIV and other viruses. However, in some cases, this approach is difficult due to the absence of a suitable system allowing infection of a cell in vitro.
In other cases, even if a suitable cell-based assay is available, this procedure may be too cumbersome or expensive. This is the case for certain dangerous viruses—such as those that require BSL-3 and/or BSL-4 facilities. Establishing a screening process for over a large amount of compounds in a BSL-3 or BSL-4 containment facility has not been achieved yet because of this heavy expense and burden. For example, flaviviruses belong to this class of viruses. These viruses require from BSL-2 to BSL-4 facilities (e.g., Dengue, WNV and/or Kyasanur Forest viruses). Thus, in such cases, it is preferable to screen potentially antiviral compounds directly on viral target proteins.
For efficiency, especially considering the difficulty with certain, more dangerous viruses, the characterization in molecular terms of the target, the viral polymerase, is of prime importance in the screening and selection of antiviral compounds. In the case of the flavivirus RNA polymerase (“NS5” or sometimes referred to herein as “NS5Pol”), this task has proven to be difficult for several reasons. First, polymerase genes have been notoriously difficult to clone in their entirety. When available, recombinant NS5 has been reported to be unstable in bacterial hosts. In addition, the notoriously low yield of soluble purified NS5 is a limiting factor to set up polymerase-activity assays. Another possible reason for the described difficulties is the fact that NS5 does not carry a single enzymatic activity.
Very recently, we described an N-terminal domain of NS5 (sometimes referred to herein as “NS5 methyltransferase domain”) which acts as an S-adenosyl-L-methionine (AdoMet)-utilizing RNA-cap 2′Omethyltransferase, thus participating in mRNA capping, which is generally understood as the process of adding a guanosine nucleotide to the 5′ end of mRNA (the methelyated end of guanosine) (Egloff & Benarroch, 2002). Additionally, we showed that the NS5 methyltransferase domain binds GTP analogues.
Due to the nature and proximity of the NS5 methyltransferase domain to the polymerase domain of the flavivirus, the description and characterization of the NS5 methyltransferase domain clearly shows that some nucleoside analogues and inhibitors of flavivirus replication could potentially be, in fact, mRNA-capping inhibitors without any effect on the polymerase activity. Likewise, it is very possible to mistakenly identify a compound as binding to NS5 and characterizing the binding data as potentially interesting for inhibition of the polymerase, but, in reality, only the RNA-capping has been affected. Therefore, it would be useful to identify and define the “junction” or sequence between the NS5 methyltransferase domain and the polymerase domain.